Techniques enabling efficient transfer of a substance of interest from the external medium into cells, and particularly to cellular nuclei, are of considerable interest in the field of biotechnology. These techniques may be useful for protein or peptide production, for regulation of gene expression, for analysis of intracellular signaling channels and for the analysis of the effect of transport of a variety of different substances into a cell (or cell nucleus). One important application of such a technique is gene therapy. However, it is limited by the inability of the gene transfer vectors to transfer the biologically active substance into the cytoplasm or nuclei of cells in the host to be treated without affecting the host genome or altering the biological properties of the active substance.
Several techniques have been developed in an effort to efficiently transfer DNA into cells. Representative examples include coprecipitating DNA with calcium phosphate or DEAE-dextran or electroporation, both of which enable DNA to penetrate the plasma membrane and then enter the cell and/or nucleus. Both of these techniques suffer from low transfer efficiency and a high percentage of cell death. Other methods employ a conjugate of a virus-related substance with a strong affinity for DNA and a nucleic acid. However, the viral conjugates are difficult to use, and there are some risks related to the use of virus components. See, e.g., U.S. Pat. No. 5,521,291. Receptor-mediated endocytosis is also widely exploited in experimental systems for the targeted delivery of therapeutic agents into cells (36). Ligand-containing complexes are either selectively internalized by receptors located in the cell membrane which are specific for the ligands, or by specific antibodies located in membrane constituents. Endocytotic activity has been described for many receptors including IgG Fc, somatostatin, insulin, IGF-I and II, transferrin, EGF, GLP-1, VLDL or integrin receptors (35;37–43).
Proprotein convertases are also an example of a cell surface receptor which gets internalized through receptor mediated endocytosis. These proteins have been shown to be responsible for conversion of precursors of peptide hormones, neuropeptides, and many other proteins into their biologically active forms. All cleavage sites for the proprotein convertase family obey to the consensus R-X-X-R. The mammalian proprotein convertases can be classified into three groups on the basis of their tissue distribution. Furin, PACE4, PC5/PC6, and LPCIPC7/PC8/SPC7 are expressed in a broad range of tissues and cell lines. In contrast, expression of PC2 and PC1/PC3 is limited to neuroendocrine tissues, such as pancreatic islets, pituitary, adrenal medulla and many brain areas. Expression of PC4 is highly restricted to testicular spermatogenic cells. The neuroendocrine-specific convertases, PC2 and PC1/PC3, are mainly localized in secretory granules. PC5/PC6A has also been reported to be localized to secretory granules. Furthermore, indirect evidence has suggested that a proportion of proprotein convertases molecules is present on the cell surface, and it has been shown that furin cycles between the TGN and the cell surface (reviewed in (1)). Taken together, these properties indicate that proprotein convertases transport extracellular ligands into the intracellular space.
The isolation of peptide sequences that direct efficient receptor-mediated endocytosis are profoundly boosted by the use of phage display technologies (44). Phage display libraries are extremely powerful tools that provide for a practically unlimited source of molecular variants including modifications of natural ligands to cell receptors (45) and short peptides (46). Similar libraries have also been injected directly into mice and peptide sequences that show a 13-fold selectivity for brain and kidney have been successfully isolated (48;49).
A need remains in the art for an efficient, non-biologically altering, low-risk means to target various cell types for the intracellular delivery of drugs and therapeutic agents. Thus, small transporter peptides that selectively target specific cell types may be derived from large phage display libraries. The advantages of small peptide carriers such as those obtained using phage display libraries include high quality and purity, low immunogenicity and the potential for highly efficient delivery to all cells in an organism (26). Accordingly, peptide carriers have the potential to improve upon conventional transporters such as liposomes or viruses for the efficient delivery of many macromolecules (see for example (50;51)).